Lung cancer is the most common form of cancer in the world. Estimates for the year 1985 indicate that there were about 900,000 cases of lung cancer worldwide. (Parkin, et el., "Estimates of the worldwide incidence of eighteen major cancers in 1985," Int J Cancer 1993; 54:594-606). For the United States alone, 1993 projections placed the number of new lung cancer cases at 170,000, with a mortality of about 88%. (Boring, et al., "Cancer statistics," CA Cancer J Clin 1993; 43:7-26). Although the occurrence of breast cancer is slightly more common in the United States, lung cancer is second behind prostate cancer for males and third behind breast and colorectal cancers for women. Yet, lung cancer is the most common cause of cancer deaths.
The World Health Organization classifies lung cancer into four major histological types: (1) squamous cell carcinoma (SCC), (2) adenocarcinoma, (3) large cell carcinoma, and (4) small cell lung carcinoma (SCLC). (The World Health Organization, "The World Health Organization histological typing of lung tumours," Am J Clin Pathol 1982; 77:123-136). However, there is a great deal of tumor heterogeneity even within the various subtypes, and it is not uncommon for lung cancer to have features of more than one morphologic subtype. The term non-small cell lung carcinoma (NSCLC) includes squamous, adenocarcinoma and large cell carcinomas.
Typically, a combination of X-ray and sputum cytology is used to diagnose lung cancer. Unfortunately, by the time a patient seeks medical help for their symptoms, the cancer is at such an advanced state it is usually incurable. Cancer Facts and Figures (based on rates from NCI SEER Program 1977-1981), New York: American Cancer Society, 1986). Routine large-scale radiologic or cytologic screening of smokers has been investigated. Studies concluded that cytomorphological screening did not significantly reduce the mortality rate from lung cancer and was not recommended for routine use. ("Early lung cancer detection: summary & conclusions," Am Rev Respir Dis 1984; 130:565-70). However, in a subpopulation of patients where the cancer is diagnosed at a very early stage and the lung is surgically resectioned, there is a 5-year survival rate of 70-90%. (Flehinger, et al., "The effect of surgical treatment on survival from early lung cancer," Chest; 1992, 101:1013-1018; Melamed, et al,. "Screening for early lung cancer: results of the Memorial Sloan-Kettering Study in New York," Chest; 1984 86:44-53). Therefore, research has focused on early detection of tumor markers before the cancer becomes clinically apparent and while the cancer is still localized and amenable to therapy.
The identification of antigens associated with lung cancer has stimulated considerable interest because of their use in screening, diagnosis, clinical management, and potential treatment of lung cancer. International workshops have attempted to classify the lung cancer antigens into 15 possible clusters that may define histologic origins. (Souhami et al., "Antigens of lung cancer: results of the second international workshop on lung cancer antigens," JNCI 1991; 83:609-612). As of 1988, more than 200 monoclonal antibodies (MAb) have been reported to react with human lung tumors. (Radosevich, et al., "Monoclonal antibody assays for lung cancer," In: Cancer Diagnosis in Vitro Using Monoclonal Antibodies. Edited by H. A. Kupchik. New York: Marcel Dekker, 1988;101-121).
MAbs for lung cancer were first developed to distinguish NSCLC from SCLC. (Mulshine, et al., "Monoclonal antibodies that distinguish nonsmall-cell from small-cell lung cancer," J Immunol 1983; 121:497-502). In most cases, the identity of the cell surface antigen with which a particular antibody reacts is not known, or has not been well characterized. (Scott, et al., "Early lung cancer detection using monoclonal antibodies," In: Lung Cancer. Edited by J. A. Roth, J. D. Cox, and W. K. Hong. Boston: Blackwell Scientific Publications, 1993:310-324).
MAbs have been used in the immunocytochemical staining of sputum samples to predict the progression of lung cancer. (Tockman, et al., "Sensitive and specific monoclonal antibody recognition of human lung cancer antigen on preserved sputum cells: a new approach to early lung cancer detection," J Clin Oncol 1988; 6:1685-1693). In the study, two MAbs were utilized, 624H12 which binds a glycolipid antigen expressed in SCLC and 703D4 which is directed to a protein antigen of NSCLC. Of the sputum specimens from participants who progressed to lung cancer, two-thirds showed positive reactivity with either the SCLC or the NSCLC MAb. In contrast, of those that did not progress to lung cancer, 35 of 40 did not react with the SCLC or NSCLC Mab. This study suggests the need for the development of additional early detection targets to discover the onset of malignancy at the earliest possible stage.
Carcinoembryonic antigen (CEA) is a frequently studied tumor marker of cancer including lung cancer. (Nutini, et al., "Serum NSE, CEA, CT, CA 15-3 levels in human lung cancer," Int J Biol Markers 1990; 5:198-202) . Squamous cell carcinoma antigen is another established serum marker. (Margolis, et al., "Serum tumor markers in non-small cell lung cancer," Cancer 1994; 73:605-609.sub.-). Other serum antigens for lung cancer include antigens recognized by MAbs 5E8, 5C7, and 1F10, the combination of which distinguishes between patients with lung cancer from those without (Schepart, et al., "Monoclonal antibody-mediated detection of lung cancer antigens in serum," Am Rev Respir Dis 1988; 138:1434-8), Furthermore, the combination of 5E8, 5C7 and 1F10 was more sensitive, specific and accurate for identifying NSCLC when compared to results from a combination of the CEA and squamous cell carcinoma antigen tests. (Margolis, et al., Cancer 1994; 73:605-609).
Serum CA 125, initially described as an ovarian cancer-associated antigen, has been investigated for its use as a prognostic factor in NSCLC. (Diez, et al., "Prognostic significance of serum CA 125 antigen assay in patients with non-small cell lung cancer," Cancer 1994; 73:1369-76). The study determined that the preoperative serum level of CA 125 antigen is inversely correlated with survival and tumor relapse in NSCLC.
Despite the numerous examples of MAb applications, none has yet emerged that has changed clinical practice. (Mulshine, et al., "Applications of monoclonal antibodies in the treatment of solid tumors," In: Biologic Therapy of Cancer. Edited by V. T. Devita, S. Hellman, and S. A. Rosenberg. Philadelphia: JB Lippincott, 1991, pp. 563-588). MAbs alone may not be the answer to early detection because there has only been moderate success with immunologic reagents for paraffin-embedded tissue. Secondly, lung cancer may express features that cannot be differentiated by antibodies; for example, chromosomal deletions, gene amplification, or translocation and alteration in enzymatic activity.
After the gene to the MAb recognized surface antigen has been cloned, cytogenetic and molecular techniques may provide powerful tools for screening, diagnosis, management and ultimately treatment of lung cancer. An example of a lung cancer antigen that has been cloned is the adenocarcinoma-associated antigen. This antigen, recognized by KS1/4 MAb, is an epithelial malignancy/epithelial tissue glycoprotein from the human lung adenocarcinoma cell line UCLA-P3. (Strnad, et al., "Molecular cloning and characterization of a human adenocarcinoma/epithelial cell surface antigen complementary DNA," Cancer Res 1989; 49:314-317). The antigen has been found on all adenocarcinoma cells tested and in various corresponding normal epithelial cells. Northern blot analysis indicated that transcription of the adenocarcinoma-associated antigen was detected in RNA isolated from normal colon but not in RNA isolated from normal lung, prostate, or liver. Therefore identification of adenocarcinoma-associated antigen in lung cells may prove to be diagnostic for adenocarcinoma.
The cloning of CEA and the nonspecific crossreacting antigen (NCA) has allowed the development of specific DNA probes which discriminate their expression in lung cancer at the mRNA level. (Hasegawa, et al., "Nonspecific crossreacting antigen (NCA) is a major member of the CEA-related gene family expressed in lung cancer," Br J Cancer 1993; 67:58-65). NCA is a component of the CEA gene family in lung cancer and is also recognized by anti-CEA antibodies, especially polyclonal antibodies. Because of the crossreactivity, investigations to analyze CEA and NCA separately in lung disease had been difficult. The use of DNA probes determined that lung cancer cells fall into three different types according to their CEA and/or NCA expression by Northern blot analysis. Specifically, lung cancers expressed both CEA and NCAmRNA, only NCAmRNA, or neither mRNA. CEA-relatedmRNA expression was always accompanied by NCAmRNA expression and there were no cases of CEAmRNA expression alone. The separate assessment of CEA and NCA expression in lung cancers may be important in determining the prognosis of lung cancers because the antigens have been described as cell-cell adhesion molecules and may play a role in cancer metastasis.
Another method to detect the presence of an antigen gene or its mRNA in specific cells or to localize an antigen gene to a specific locus on a chromosome is in situ hybridization. In situ hybridization uses nucleic acid probes that recognize either repetitive sequences on a chromosome or sequences along the whole chromosome length or chromosome segments. By tagging the probes with radioisotopes or color detection systems, chromosome regions can be identified within the cell. Investigations using in situ hybridization have demonstrated numerous chromosomal abnormalities in samples from human tumors, including bladder, neuroectodermal, breast, gastric and lung cancer tumors. (Kim, et al., "Interphase cytogenetics in paraffin sections of lung tumors by non-isotopic in situ hybridization. Mapping Genotype/phenotype heterogeneity," Am J Pathol 1993; 142:307-317).
Fluorescent in situ hybridization (FISH) allows cells to be stained so that genetic aberrations resulting in changes in gene copy number or structure can be quantitated by fluorescent microscopy. In this technique, a chemically labeled single-stranded nucleic acid probe homologous to the target nucleic acid sequence is annealed to denatured nucleic acid contained in target cells. The cells may be mounted on a microscope slide, in suspension or prepared from paraffin-embedded material. Treating the chemically modified probes with a fluorescent ligand makes the bound probe visible. FISH has been used for (1) detection of changes in gene copy number and gene structure; (2) detection of genetic changes, even in low frequency subpopulations; and (3) detection and measurement of the frequency of residual malignant cells. (Gray, et al., "Molecular cytogenetics in human cancer diagnosis," Cancer 1992; 69:1536-1542).
Other molecular markers for lung cancer include oncogenes and tumor suppressor genes. Dominant oncogenes are activated by mutation and lead to deregulated cellular growth. Such genes code for proteins that function as growth factors, growth factor receptors, signal transducing proteins and nuclear proteins involved in transcriptional regulation. Amplification, mutation, and translocations have been documented in many different cancer cells and have been shown to lead to gene activation or overexpression.
The ras family of oncogenes comprises a group of membrane associated GTP-binding proteins thought to be involved in signal transduction. Mutations within the ras oncogenes, resulting in sustained growth stimulation, have been identified in 15 to 30% of human NSCLC. (Birrer, et al., "Application of molecular genetics to the early diagnosis and screening of lung cancer," Cancer 1992; 52suppl; 2658s-2664s). Patients with tumors containing ras mutations had decreased survival compared with patients whose tumors had no ras mutations. Polymerase chain reaction (PCR) amplification of ras genes can be analyzed to determine the presence of mutations by several methods: (a) differential hybridization of .sup.32 P-labeled mutated oligonucleotides; (b) identification of new restriction enzyme sites created by the activating mutation; (c) single-strand conformational polymorphisms; and (d) nucleic acid sequencing. These methods combined with PCR technology could allow detection of an activated ras gene from sputum specimens.
Another family of dominant oncogenes, the erb B family, has been found to be abnormally expressed in lung cancer cells. This group codes for membrane-associated tyrosine kinase proteins and contains erb B1, the gene coding for the epidermal growth factor (EGF) receptor, and erb B2 (also called Her-2/neu). The erb B1 gene has been found to be amplified in NSCLC (up to 20% of squamous cell tumors), while the EGF receptor has been shown to be overexpressed in many NSCLC cells (approximately 90% of squamous cell tumors, 20 to 75% of adenocarcinomas, and rarely in large cell or undifferentiated tumors). (Birrer, et al., Cancer 1992:52 suppl; 2658s-2664s). Amplification of the related oncogene erb B2 (Her-2/neu) occurs infrequently in lung cancer but is a negative prognostic factor in breast cancer. However, overexpression of the erb B2 protein product, p185.sup.neu, occurs in some NSCLC and may be related to poor prognosis. (Kern, et al., "p185.sup.neu expression in human lung adenocarcinomas predicts shortened survival," Cancer Res 1990; 50:5184-5191).
A third family of dominant oncogenes involved in lung cancer is the myc family. These genes encode nuclear phosphoproteins, which have potent effects on cell growth and which function as transcriptional regulators. Unlike ras genes, which are activated by point mutations in lung cancer cells, the myc genes are activated by overexpression of the cellular myc genes, either by gene amplification or by rearrangements, each ultimately leading to increased levels of myc protein. Amplification of the normal myc genes is seen frequently in SCLC and rarely in NSCLC.
The loss or inactivation of tumor suppressor genes may also be important steps in the pathway leading to invasive cancer. Tumor suppressor genes function normally to suppress cellular proliferation, and since they are recessive oncogenes, mutations or deletions must occur in both alleles of these genes before transformation occurs.
A phosphoprotein p53, which is encoded by a gene located on chromosome 17p, suppresses transformation in its wild-type state. While in its mutant state, p53 acts as a dominant oncogene. p53 functions in DNA binding and transcription activation. Mutations of p53 have been found in many human cancers including colon, breast, brain and lung cancer cells. (Birrer, et al., Cancer Res.(suppl) 1992, 52:2658s-2664s). In NSCLC cell lines, p53 mutations have been found at a rate of up to 74%. (Mitsudomi, et al., "p53 gene mutations in non-small-cell lung cancer cell lines and their correlation with the presence of ras mutations and clinical features," Oncogene 1992; 7:171-180).
Despite all of the advances made in the area of lung cancer, medical and surgical intervention has resulted in little change in the 5-year survival rate for lung cancer patients. Early detection holds the greatest hope for successful intervention. There remains a need for a practical method to diagnose lung cancer as close to its inception as possible. In order for early detection to be feasible, it is important that specific markers be found and their sequences elucidated.
A lung cancer marker antigen, specific for NSCLC, has now been found, sequenced, and cloned. The antigen is useful in methods for detection of non-small cell lung cancer and for potential production of antibodies and probes for treatment compositions.